1. Field of the Invention
The present invention relates to an amidase enzyme, nucleic acids encoding the amidase, as well as methods of employing the nucleic acids and/or amidase to produce, for example, enantiomerically enriched compounds such as D-amino acids.
2. Discussion of the Background
Amidases or amidohydrolases are classified according to the E.C. system into two sub-classes, E.C. 3.5.1.1 to 3.5.1.77 and E.C. 3.5.2.1 to 3.5.2.14. Representatives of the first sub-class are e.g. asparaginase (E.C. 3.5.1.1), urease (E.C. 3.5.1.5) and the acylamide amidohydrolase considered in more detail here (E.C. 3.5.1.4).
The acylamide amidohydrolase is widespread within microorganisms and occurs, inter alia, in species such as Corynebacteria, Pseudomonas, Bacilli, Brevibacteria, Rhodococci and Alcaligenes. These are usually inducible enzymes, the specificity of which varies greatly from organism to organism (Maestracci, M.; Bui, K.; Thiéry, A.; Arnaud, A.; Galzy, P. (1988), The Amidases from a Brevibacterium Strain: Study and Applications, Adv. Biochem. Eng. 36, 69–115).
The enzymes from Mycobacterium neoaurum ATCC 25795 (Hermes, H. F. M.; Tandler, R. F.; Sonke, T.; Dijkhuizen, L.; Meijer, E. M. (1994), Purification and Characterization of an L-Amino Amidase from Mycobacterium neoaurum ATCC 25795, Appl. Environ. Microbiol. 60, 153–159) and Pseudomonas putida ATCC 12633 (Hermes, H. F. M.; Sonke, T.; Peters, P. J. H.; van Balken, J, A. M.; Kamphuis, J.; Dijkhuizen, L.; Meijer, E. M. (1993), Purification and Characterization of an L-Aminopeptidase from Pseudomonas putida ATCC 12633, Appl. Environ. Microbiol. 59, 4330–4334) are of particular industrial importance for the hydrolysis of L-amino acid amides. Both enzymes show a relatively high affinity for N-branched amino acid amides or dipeptides and are therefore classified as aminopeptidases (E.C. 3.4.). The L-aminopeptidase from Pseudomonas putida ATCC 12633 is employed for stereospecific cleavage of a D,L-phenylglycinamide mixture into D-phenylglycinamide and L-phenylglycine. A process developed by the DSM utilizes whole cells of Pseudomonas putida for the preparation of pure D- and L-amino acids from D,L-amino acid amides (Kamphuis, J.; Boesten, W. H. J.; Broxterman, Q. B.; Hermes, H. F. M.; Balkan van, J. A. M.; Meijer, E. M.; Shoemaker, H. E. (1990), New developments in the chemo-enzymatic production of amino acids, Adv. Biochem. Eng. Biotechnol. 42, 133–186).
A process for the preparation of L-amino acids and amino acid amides from D,L-α-aminonitriles is described in Klages, U.; Weber, A. (1988), Verfahren zur Herstellung von L-Aminosäuren und Aminosäureamiden, DE 3 816 063 A1; WO 8 910 969). In this biotransformation with whole cells, D,L-aminonitriles are first hydrolysed to D,L-amino acid amides with Acinetobacter calcoaceticus DSM 3875. Complete conversion into the L-amino acid is in principle possible with an L-amino acid amidase and an amino acid amide racemase in Arthrobacter sp. ATCC 31652 or Corynebacterium sp. ATCC 31662.
As a result of the discovery of amino acid amide racemases in Pseudomonas putida and Rhodococcus sp., a process for the racemization of amino acid amides and hydrolysis by an L- or D-amidase to give the corresponding amino acid is described in Godtfredsen, S. E.; Clausen, K.; Ingvorsen, K.; Hermes, H. F.; Van Balken, J. A.; Meijer, E. M. (1989), EP 0 307 023; WO 8 901 525. A D-amidase activity has been described here in Pseudomonas putida NCIB 40042 and Rhodococcus sp. NCIB 40041.
The discovery of a further amino acid racemase in Klebsiella oxytoca is described by Hermes, H. F. M.; Peeters, W. P.; Peters, P. J. (1990), EP 0 383 403.
Amidases with a D-specificity to amino acid amides have been described in Comamonas acidovorans KPO-2771–4 (Hayashi, T.; Yamamoto, K.; Matsuo, A.; Otsubo, K.; Muramatsu, S.; Matsuda, A.; Komatsu, K.-I. (1997), Characterization and Cloning of an Enantioselective Amidase from Comamonas acidovorans KPO-2771–4, J. Ferment. Bioeng. 83, 139–145) and in two strains of Ochrobactrum anthropi, SCRC C1-38 (Asano, Y.; Kato, Y.; Yamada, A.; Kondo, K. (1992) Structural Similarity of D-Aminopeptidase to Carboxypeptidase DD and β-Lactamases, Biochem. 31, 2316–2328; Asano, Y.; Nakazawa, A.; Kato, Y.; Kondo, K. (1989), Properties of a Novel D-Stereospecific Aminopeptidase from Ochrobactrum anthropi, J. Biol. Chem. 264, 14233–14239) and SCRC-SV3 (Komeda, H. and Asano, Y. (2000), Gene cloning, nucleotide sequencing, and purification and characterisation of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3, Eur. J. Biochem. 267, 2028–2035; Asano, Y.; Mori, T.; Hanamoto, S.; Kato, Y.; Nakazawa, A. (1989), A New D-Stereospecific Amino Acid Amidase From Ochrobactrum anthropi, Biochem. Biophys. Res. Commun. 162, 470–474).
Nevertheless, there is still a need for D-amidases, especially since their substrate spectra are not covered 100% and for the preparation of poorly convertible substrates on an industrial scale under economically advantageous conditions.